The cytoplasmic replicative cycle of poliovirus, a positive-strand RNA Virus, inc u es t e 10,000-fold amplification of positive-strand RNA and drastic membrane rearrangement within the infected primate cell. To address several unresolved aspects of this replicative cycle, a well-characterized collection of conditionally lethal alleles of the poliovirus polymerase will be valuable tools. To this end, a battery of temperature-sensitive mutants in the coding region, 3D, of the viral polymerase, constructed by charged-cluster-to-alanine mutagenesis, will be characterized with respect to positive-strand synthesis, negative-strand synthesis, RNA recombination frequency, and temperature-dependence of replication in a cell-free assay. We will determine by second-site suppression analysis whether other viral proteins can interact with the mutant polymerase to rectify the primary defect. "Interaction traps" in yeast will be employed as another method to determine both cellular and viral proteins interact with 3D and the protease-polymerase fusion protein 3CD; both wild-type and mutant 3D alleles will be studied. Any cellular proteins that interact with 3D and 3CD (3Di and 3CDi proteins) will by expressed and purified from bacteria. Purified 3Di and 3CDi proteins and antibody reagents will be used to test the function of these proteins in poliovirus replication. Viral RNA replication complexes are specifically associated with large numbers of membranous vesicles that accumulate within the cytoplasm of poliovirus-infected cells. The 106-fold inhibition of poliovirus replication by brefeldin A, a drug that inhibits the formation of vesicles required for ER-to-Golgi protein traffic, suggested that the proliferating vesicles derive directly from the ER or Golgi or both. By expressing VSV G protein in a cap-independent fashion in poliovirus-infected cells, we discovered that protein transport is disturbed during infection, proceeding no further than the cis-Golgi. We will characterize this inhibition of protein secretion in poliovirus-infected cells by monitoring transiently- expressed VSV-G protein, by characterizing the vesicles that accumulate during poliovirus infection, and by determining which viral protein or proteins are responsible for the secretion block By studying the mechanism of viral subversion of subcellular vesicles, we will learn more about the protein secretion apparatus of the host cell as well as the curious association of poliovirus replication complexes with what are, we propose, normal transport vesicles within the cell.